This project will investigate the genetic and regulatory functions of the mammalian cell cytoplasm. Mutants of mouse L cells and human HeLa cells will be sought which are resistant to inhibitors of mitochondrial protein synthesis or oxidative phosphorylation. These will be tested for cytoplasmic inheritance of their resistance characteristics by the methods established in this laboratory for the cytoplasmically inherited chloramphenicol-resistant (CAP R) mutants. The molecular entity altered by each cytoplasmic mutation will be investigated. Bifactorial recombination experiments between chloramphenicol- and carbomycin-resistant mutants will be performed in both cis and trans configurations. The same crosses with one parent enucleated will detect any nuclear influences on cytoplasmic genetic behavior. All crosses can be intraspecific or interspecific, and can be between cells of similar or different tissue origin. These techniques can be extended to multifactorial crosses. The regulation of tyrosine aminotransferase (TAT) in rat hepatoma cells (HTC) is being studied. Cytoplasmically labeled enucleated CAP R L cells will be fused to H3-thymidine labeled HTC cells, and the hybrids assayed for TAT activity. If cytoplasmic repression of TAT occurs, the nature, synthesis, and specificity of the cytoplasmic repressor will be examined.